In Vivo Fluorescence Imaging to Localize Antibodies in a Mouse Tumor Xenograft Model

1. Expression and purification of antibodies

1. Maintenance of CHO cells
   1. Grow CHO cells in FreeStyle CHO Media supplemented with commercially available 1x glutamine supplement at 37 ˚C shaking at 130 rpm with 5% CO₂ using delong Erlenmeyer flasks, either glass or disposable.
      NOTE: It is highly recommended to use a baffled flask with a vented cap for increased agitation and to improve gas transfer during shaking conditions. Significantly reduced antibody yield has been obtained with regular flasks (non-baffled) due to limited agitation of the suspension culture.
   2. Maintain the cell number between 1-5 x 10⁶ cells/mL with >95% cell viability. If the cell number increases over 5 x 10⁶ cells/mL, split the cells. Never allow CHO cells to reach below 0.2 x 10⁶ cells/mL.
2. Transfection of CHO cells
   1. Grow CHO cells in 200 mL of media (2 x 10⁶ cells/mL) in delong Erlenmeyer baffled flasks. 
      NOTE: Suspension cultures grown in baffled flasks have always produced higher protein yields than when grown in non-baffled flasks.
   2. In a 15 mL tube, take 5 mL of CHO FreeStyle media and add 50 μg of VH clone DNA and 75 μg of VL clone DNA. Vortex to mix well.
   3. Incubate the DNA mixture at room temperature for 5 min.
      NOTE: Longer incubation reduces the protein yields.
   4. Add 750 μL of 1 mg/mL polyethyleneimine (PEI) stock to the DNA solution and aggressively vortex the mixture for 30 s. Incubate at room temperature for an additional 5 min.        
      NOTE: PEI must be made fresh. Multiple freeze-thaw cycle of PEI reduces overall yield significantly.
   5. Add the entire mixture of DNA and PEI on the cells while manually shaking the flask. Immediately incubate the delong Erlenmeyer baffled flasks with cells at 37 ˚C shaking at 130 rpm.
3. Expression
   NOTE: The antibody has secretory signal peptide engineered to its N-terminal end, which helps antibodies to be secreted out into the media.
   1. Grow the transfected cells at 37 ˚C, with shaking at 130 rpm on the Day 1.
   2. On the Day 2, add 2 mL of 100x anti-clumping agent and 2 mL of 100x anti-bacterial-anti-mycotic solution. Shift the flask to lower temperature (32-34 ˚C), with shaking at 130 rpm.
   3. On every fifth day, add 10 mL of Tryptone N1 feed, and 2 mL of 100x glutamine supplement.
   4. Keep counting the cells every third day using hemocytometer after staining an aliquot of cells with trypan blue stain. Ensure that the cell viability stay above 80%.
   5. On the Day 10 or 11, harvest the medium for antibody purification. Spin the culture at 3000 x g, 4 ˚C for 40-60 min and then filter the clear media using 0.22 µM bottle filters.
4. Purification
   NOTE: Antibody purification is performed using commercially available Protein-A column (see Table of Materials), using a peristaltic pump.
   1. Equilibrate the column with two column volume of binding buffer (20 mM sodium phosphate at pH 7.4).
   2. Pass the filtered media containing antibody (obtained in step 1.3.5) through the column at the flow rate of 1 mL/min.
   3. Wash the column with two-column volume of binding buffer.
   4. Elute the antibody into 500 μL fractions using 5 mL elution buffer (30 mM sodium acetate at pH 3.4).
   5. Neutralize the pH of the eluted antibody by adding 10 μL of neutralization buffer (3 M sodium acetate at pH 9) per fraction.
      NOTE: Fraction number 3-6 contains most of the antibody. It is advisable to keep all the fractions in case the antibody is eluted in a later fraction than expected.
   6. Measure the concentration of purified antibody using a spectrophotometer by selecting the default protocol for IgG. The final concentration of the antibody is obtained in mg/mL by considering the molecular weight and absorption coefficient.

2. Fluorescent labeling

NOTE: Antibodies are labeled with the infrared dye that contains an NHS ester reactive group, which couples to proteins and form a stable conjugate. This reaction is pH sensitive and works best at pH 8.5. Fluorescent conjugates labeled with the dye display an absorption maximum of 774 nm, and an emission maximum of 789 nm. pH 8.5 is key for effective conjugation.

1. Dialyze 0.5 mL of the antibody using a dialysis cassette (0.1-0.5 mL) in 1 L of conjugation buffer (50 mM phosphate buffer at pH 8.5). After 4 h transfer the dialysis cassette to fresh buffer and dialyze overnight.
2. Setup a conjugation reaction by adding 0.03 mg of IRDye 800CW per 1 mg of antibody in a reaction volume of 500 μL.
   NOTE: IRDye 800CW is dissolved in DMSO at a concentration of 10 mg/mL.
3. Carry out labeling reactions for 2 h at 20 °C.    
   NOTE: Increasing time beyond 2 h does not improve the labeling.
4. Purify labeled conjugates by extensive dialysis against 1x PBS.
5. Estimate the degree of labeling by measuring the absorbance of the dye at 780 nm and absorbance of the protein at 280 nm. The dye contribution to the 280 nm signal is 3%.
6. Calculate the dye/protein ratio using this formula:
   
   where 0.03 is a correction factor for the absorbance of the dye used at 280 nm (equal to 3.0 % of its absorbance at 780 nm), ε_Dye and ε_Protein are molar extinction coefficients for the dye and the protein (antibody) respectively.
   NOTE: ε_Dye is 270,000 M^-1 cm^-1 and ε_Protein is 203,000 M^-1 cm^-1 (for a typical IgG) in 1:1 mixture of PBS: methanol. Proteins other than IgG may have very different molar extinction coefficients. Use of correct extinction coefficient for the protein of interest is essential for accurate determination of D/P ratio.
7. Calculate the final protein concentration using this formula:
   
   NOTE: Always confirm the antigen-binding efficacy of labeled and unlabeled antibody using ELISA (Enzyme Linked Immunosorbent Assay) or flow cytometry before proceeding to in vivo studies.

3. Mouse xenograft studies

1. Preparation of tumor cells for injection
   1. Grow OVCAR-3 cells in RPMI-1640 Medium, supplemented with 10% of FBS and 1x penicillin-streptomycin.
   2. The day before the injection, subculture cells into new 100 mm culture dishes with 10 mL of complete medium/dish. Use cell number of 0.5-1 x 10⁶ cells/dish.
   3. Incubate cultures at 37 °C temperature, 95% humidity and 5% CO₂ for 20-24 h.
   4. On the day of injection, remove the growth medium from culture dishes. Thoroughly rinse the cell layer with Ca²⁺/Mg²⁺ free Dulbecco's phosphate-buffered saline (DPBS) to remove dead cells, cellular debris and all traces of serum, which may interfere in trypsin action.
   5. Trypsinize the cells by adding 1.0-1.5 mL of Trypsin-EDTA solution to each dish and try to spread the solution by tilting the dish all around followed by incubation at 37 °C for 5-10 min. 
      NOTE: Observe cells under an inverted microscope to check the actual trypsinization status. Under trypsinization results in lower number of cell detachment from the surface of culture dish, over trypsinization induces cellular stress. So, proper trypsinization is important.
   6. Add 1.0-1.5 mL of complete growth medium to each dish to stop the trypsin action, after that re-suspend cells by pipetting gently.         
      NOTE: Gentle pipetting is important to maintain cell health.
   7. Collect the cell suspension into a 15 mL conical tube and spin at 250 x g for 5 min at room temperature.
   8. Collect the cell pellet after removing the supernatant and wash the cells by re-suspending the pellet in 1x DPBS.
   9. Spin the cell suspension at low speed 250 x g for 5 min.
   10. Add 500 µL of DPBS and re-suspend cells by gentle pipetting to get single cell suspension.
   11. Count cells using hemocytometer or automated cell counter.
       NOTE: For better accuracy, repeat the counting for three times and take an average.
   12. Adjust the volume in such a way so that the final cell density will be 1 x 10⁸ cells/mL.
2. Subcutaneous injection of cells to develop mouse xenografts
   NOTE: All procedures should be done in a BSL2 safety cabinet. Athymic Nude Foxn1^nu/Foxn1⁺ mice have been used in the current study.
   1. Take 50 µL of the cell suspension into a 1.5 mL tube and mix with 50 µL of basement membrane matrix medium.
      NOTE: Basement membrane matrix medium tends to form a gel like state at room temperature, so carefully maintain the cells and the matrix medium mixture on ice. It is advisable to keep tubes, tips and syringes in the fridge and then transfer on ice prior to the animal injection.
   2. Agitate the mixture to avoid any cell clumping. Then, take this 100 µL of the cell suspension-matrix medium mixture that contains 5 x 10⁶ cells, into a 1 cc syringe.
   3. Gently lift the skin of the animal to separate the skin from the underlying muscle layer and slowly inject the cell suspension (100 µL) under the skin (5 x 10⁶ cells), with a 26 G needle. Wait for a few seconds before taking the needle out, so that basement matrix medium can form the semi-solid gel like structure along with cells under the skin, preventing the mixture coming out from the site of injection.
      NOTE: Cells needs to be tested prior to injection for any contamination which may harm to immunodeficient mice. While injecting do not put the needle too deep into the skin as this may form the tumor deeper than expected.
   4. Keep the animal in a sterile cage and observe for around 20 min.
   5. Observe the mice for 2-3 weeks and allow the tumor to grow up to 500 mm³ size.

4. Antibody localization using an in vivo imaging system

NOTE: In vivo, imaging equipment (see Table of Materials) used in this experiment uses a set of high-efficiency filters and spectral un-mixing algorithms for noninvasive visualization and tracking of cellular and genetic activity within a living organism in real-time. The system provides both fluorescence and bioluminescence monitoring capability.

1. Inject 25 μg of dye-labeled antibody via the tail vein.
   1. Anesthetize tumor-bearing mice using 2% isoflurane. Check for the lack of response to pedal reflexes.
   2. Once mice stop moving, dilate the lateral tail vein by applying worm water.
   3. Inject 25 μg (in 100 μL) of labeled antibody using a 1 cc insulin syringe with a 26 G needle.
   4. Similarly, as a negative control, label and inject the non-specific IgG1 Isotype antibody that does not target cancer cells.
2. Perform in vivo live imaging after 8, 24, 48 h, etc. of antibody injections.
   1. In the associated software, click Initialize, located in the control panel and confirm that the stage temperature is 37 °C.
   2. Turn on the oxygen supply, all the pumps on the anesthesia system, isoflurane gas supply to anesthetic chamber, and set the isoflurane vaporizer valve to 2%.
   3. Transfer the mice to the anesthetic chamber and wait till the mice are completely anesthetized. Apply the eye lubricating ointment to avoid drying of their eyes.
   4. Go to the Control panel, set up the fluorescence imaging through the Imaging Wizard option, and select the excitation at 773 nm and emission at 792 nm.   
      NOTE: The default auto exposure settings provide a good fluorescent image. However, the auto exposure preferences can be modified as per the needs.
   5. Transfer the anesthetized mice into the imaging chamber and assemble it on the imaging field using a nose cone. The imaging stage provides the option to accommodate 5 mice at a time. 
      NOTE: Have control mice imaged along with the test mice to have a similar amount of exposure and other settings during analysis.
   6. Once everything is ready, select the Acquire option on the control panel for the image acquisition.
   7. With Autoexposure settings the system generates the image within a minute. The generated image is the overlay of fluorescence on photographic image with optical fluorescence intensity displayed in units of counts or photons, or in terms of efficiency.